1. Field of the Invention
The present invention relates generally to a method and compound for sensing the presence of anionic phospholipids, especially phosphatidylserine, on the surface of vesicles and cells.
2. Description of the Prior Art
The asymmetric transmembrane distribution of phospholipids is a fundamental feature of normal cell operation. In the case of animal plasma membranes, most of the phosphatidylethanolamine (PE) and phosphatidylserine (PS) is sequestered in the inner monolayer of the membrane; whereas, most of the phosphatidylcholine (PC) and sphingomyelin (SM) is in the membrane outer monolayer, see Boon, J. M. & Smith, B. D. (2002) Med. Res. Rev., 22, 251–81, the entire contents and disclosure of which is hereby incorporated by reference. This organization of lipids is generated and maintained by the concerted action of a number of phospholipid translocases that vary in lipid specificity, energy requirements, and direction of translocation, see Daleke, D. L. & Lyles, J. V. (2000) Biochim. Biophys. Acta, 1486, 108–27, the entire contents and disclosure of which is hereby incorporated by reference. The appearance of PS on the cell surface is one of the hallmarks of the early/intermediate stages of cell apoptosis and is believed to occur prior to DNA fragmentation, morphological changes, and plasma membrane permeabilization, see Schlegel, R. A. & Williamson, P. (2001) Cell Death Differ., 8, 551–63; van Heerde, W. L., Robert-Offerman, S., Dumont, E., Hofstra, L., Doevendans, P. A., Smits, J. F., Daemen, M. J. & Reutelingsperger, C. P. (2000) Cardiovasc. Res., 45, 549–59; and Williamson, P., van den Eijnde, S. & Schlegel, R. A. (2001) Meth. Cell Biol., 66, 339–64, the entire contents and disclosures of which are hereby incorporated by reference. The clearest assessment of cell apoptosis is gained from microscopy or flow cytometry analyses that examine large cell populations and identify and quantify sub-populations. A common method of detecting PS on a cell surface is to use the PS-binding protein annexin V, see van Heerde, W. L., Robert-Offerman, S., Dumont, E., Hofstra, L., Doevendans, P. A., Smits, J. F., Daemen, M. J. & Reutelingsperger, C. P. (2000) Cardiovasc. Res., 45, 549–59; Williamson, P., van den Eijnde, S. & Schlegel, R. A. (2001) Meth. Cell Biol., 66, 339–64; van Engeland, M., Nieland, L. J., Ramaekers, F. C., Schutte, B. & Reutelingsperger, C. P. (1998) Cytometry, 31, 1–9, the entire contents and disclosures of which are hereby incorporated by reference. In the presence of sufficient calcium, this 35 Kd protein has a strong affinity for membranes that are rich in negatively charged phospholipids, see Meers, P. & Mealy, T (1993) Biochemistry, 32:11711, the entire contents and disclosures of which are hereby incorporated by reference. In particular, the association constant for annexin V binding to membranes that are rich in PS is around 108 M−1, see Meers P. et al. (1991) Biochemistry, 30:2903, the entire contents and disclosure of which is hereby incorporated by reference. The utility of annexin V is derived from its selective affinity for cell plasma membranes that have externalized phosphatidylserine (PS), see van Engeland, M., Nieland, L. J., Ramaekers, F. C., Schutte, B. & Reutelingsperger, C. P. (1998) Cytometry, 31, 1–9, the entire contents and disclosure of which is hereby incorporated by reference. Annexin V is labeled with a probe, typically a fluorescent dye such as fluorescein, to allow detection by flow cytometry or an imaging method such as fluorescence microscopy. Although annexin V is a useful reagent, it is a protein with some technical drawbacks. It is susceptible to degradation and thus requires careful storage. Another concern is that up to 2.5 mM of extracellular Ca2+ may be needed for complete membrane binding, see Williamson, P., van den Eijnde, S. & Schlegel, R. A. (2001) Meth. Cell Biol., 66, 339–64, the entire contents and disclosure of which is hereby incorporated by reference. This may lead to false positive results because most animal cells have a Ca2+ dependent scramblase that may move PS to the cell surface (concentration for half maximal activation of the scramblase is 25–100 μM), see Kamp, D., Sieberg, T. & Haest, C. W. (2001) Biochemistry, 40, 9438–46, the entire contents and disclosure of which is hereby incorporated by reference. In addition, annexin V is known to associate with membrane surfaces that contain the byproducts of lipid peroxidation, see Balasubramanian, K., Bevers, E. M., Williams, G. M. & Schroit, A. J. (2001) Biochemistry, 40, 8672–8676, the entire contents and disclosure of which is hereby incorporated by reference. Furthermore, complete annexin V binding requires incubation times of up to one hour, which is problematic for kinetic assays, Zweifach, A. (2000) Biochem. J., 349, 255–60, and Williamson, P., van den Eijnde, S. & Schlegel, R. A. (2001) Meth. Cell Biol., 66, 339–64, the entire contents and disclosures of which are hereby incorporated by reference. In summary, dye labeled annexin V is used extensively as a PS-sensing reagent, but there is a need for a more stable, low-molecular-weight mimic that can sense PS-rich membranes rapidly and independently of Ca2+ levels.
The idea that a small molecule, instead of a large protein such as annexin V, may be used to detect membrane changes, such as those due to cell apoptosis, is supported by work with the fluorescent dyes MC540 and FM1-43, Laakko, T., King, L. & Fraker, P. (2002) J. Immunol. Meth., 261, 129–39, and Zweifach, A. (2000) Biochem. J., 349, 255–60, the entire contents and disclosures of which are hereby incorporated by reference. The binding of MC540 to membranes is sensitive to membrane composition and increases with apoptotic cells, see Williamson, P., van den Eijnde, S. & Schlegel, R. A. (2001) Meth. Cell Biol., 66, 339–64, the entire contents and disclosures of which are hereby incorporated by reference. However, there are disadvantages with MC540. The signal difference between normal cells and apoptotic cells is only about five fold (it is up to 100-fold with annexin V); furthermore, the dye is phototoxic. Likewise, FM1-43 is a cationic dye that binds more tightly to membranes that are rich in anionic phospholipids and gives a signal difference between normal and apoptotic cells of 6–10 fold. A problem with FM1-43 is its broad emission (515–595 nm), which makes it difficult to use a second fluorophore that emits in the fluorescein or rhodamine channels.